1.
BRENDA
–
BRENDA is an information system representing one of the most comprehensive enzyme repositories. It is a resource that comprises molecular and biochemical information on enzymes that have been classified by the IUBMB. Every classified enzyme is characterized with respect to its catalyzed biochemical reaction, kinetic properties of the corresponding reactants are described in detail. BRENDA contains enzyme-specific data manually extracted from scientific literature and additional data derived from automatic information retrieval methods such as text mining. It provides a user interface that allows a convenient and sophisticated access to the data. BRENDA was founded in 1987 at the former German National Research Centre for Biotechnology in Braunschweig and was published as a series of books. Its name was originally an acronym for the Braunschweig Enzyme Database, from 1996 to 2007, BRENDA was located at the University of Cologne. There, BRENDA developed into a publicly accessible enzyme information system, in 2007, BRENDA returned to Braunschweig. Currently, BRENDA is maintained and further developed at the Department of Bioinformatics, a major update of the data in BRENDA is performed twice a year. Besides the upgrade of its content, improvements of the interface are also incorporated into the BRENDA database. The latest update was performed in January 2015, Database, The database contains more than 40 data fields with enzyme-specific information on more than 7000 EC numbers that are classified according to the IUBMB. Currently, BRENDA contains manually annotated data from over 140,000 different scientific articles, each enzyme entry is clearly linked to at least one literature reference, to its source organism, and, where available, to the protein sequence of the enzyme. An important part of BRENDA represent the more than 110,000 enzyme ligands, the term ligand is used in this context to all low molecular weight compounds which interact with enzymes. These include not only metabolites of primary metabolism, co-substrates or cofactors, the origin of these molecules ranges from naturally occurring antibiotics to synthetic compounds that have been synthesized for the development of drugs or pesticides. Furthermore, cross-references to external resources such as sequence and 3D-structure databases. Extensions, Since 2006, the data in BRENDA is supplemented with information extracted from the literature by a co-occurrence based text mining approach. For this purpose, four text-mining repositories FRENDA, AMENDA, DRENDA and KENDA were introduced and these text-mining results were derived from the titles and abstracts of all articles in the literature database PubMed. Data access, There are several tools to access to the data in BRENDA

2.
MetaCyc
–
The MetaCyc database contains extensive information on metabolic pathways and enzymes from many organisms. MetaCyc is also used in engineering and metabolomics research. MetaCyc contains extensive data on individual enzymes, describing their subunit structure, cofactors, activators and inhibitors, substrate specificity, MetaCyc data on reactions includes predicted atom mappings that describe the correspondence between atoms in the reactant compounds and the product compounds. It also provides enzyme mini-reviews and literature references, MetaCyc data on metabolites includes chemical structures, predicted Gibbs free energies of formation, and links to external databases

3.
Protein Data Bank
–
The Protein Data Bank is a crystallographic database for the three-dimensional structural data of large biological molecules, such as proteins and nucleic acids. The PDB is overseen by a called the Worldwide Protein Data Bank. The PDB is a key resource in areas of structural biology, most major scientific journals, and some funding agencies, now require scientists to submit their structure data to the PDB. Many other databases use protein structures deposited in the PDB, for example, SCOP and CATH classify protein structures, while PDBsum provides a graphic overview of PDB entries using information from other sources, such as Gene ontology. By 1971, one of Meyers programs, SEARCH, enabled researchers to access information from the database to study protein structures offline. SEARCH was instrumental in enabling networking, thus marking the beginning of the PDB. Upon Hamiltons death in 1973, Tom Koeztle took over direction of the PDB for the subsequent 20 years, in January 1994, Joel Sussman of Israels Weizmann Institute of Science was appointed head of the PDB. In October 1998, the PDB was transferred to the Research Collaboratory for Structural Bioinformatics, the new director was Helen M. Berman of Rutgers University. In 2003, with the formation of the wwPDB, the PDB became an international organization, the founding members are PDBe, RCSB, and PDBj. Each of the four members of wwPDB can act as deposition, data processing, the data processing refers to the fact that wwPDB staff review and annotate each submitted entry. The data are automatically checked for plausibility. The PDB database is updated weekly, likewise, the PDB holdings list is also updated weekly. As of 14 March 2017, the breakdown of current holdings is as follows,103,514 structures in the PDB have a structure factor file,9,057 structures have an NMR restraint file. 2,826 structures in the PDB have a chemical shifts file, therefore, the final conformation of the protein is obtained, in the latter case, by solving a distance geometry problem. A few proteins are determined by cryo-electron microscopy, the significance of the structure factor files, mentioned above, is that, for PDB structures determined by X-ray diffraction that have a structure file, the electron density map may be viewed. The data of such structures is stored on the electron density server, however, since 2007, the rate of accumulation of new protein structures appears to have plateaued. The file format used by the PDB was called the PDB file format. This original format was restricted by the width of computer punch cards to 80 characters per line, around 1996, the macromolecular Crystallographic Information file format, mmCIF, which is an extension of the CIF format started to be phased in

4.
PubMed
–
PubMed is a free search engine accessing primarily the MEDLINE database of references and abstracts on life sciences and biomedical topics. The United States National Library of Medicine at the National Institutes of Health maintains the database as part of the Entrez system of information retrieval, from 1971 to 1997, MEDLINE online access to the MEDLARS Online computerized database primarily had been through institutional facilities, such as university libraries. PubMed, first released in January 1996, ushered in the era of private, free, home-, the PubMed system was offered free to the public in June 1997, when MEDLINE searches via the Web were demonstrated, in a ceremony, by Vice President Al Gore. Information about the journals indexed in MEDLINE, and available through PubMed, is found in the NLM Catalog. As of 5 January 2017, PubMed has more than 26.8 million records going back to 1966, selectively to the year 1865, and very selectively to 1809, about 500,000 new records are added each year. As of the date,13.1 million of PubMeds records are listed with their abstracts. In 2016, NLM changed the system so that publishers will be able to directly correct typos. Simple searches on PubMed can be carried out by entering key aspects of a subject into PubMeds search window, when a journal article is indexed, numerous article parameters are extracted and stored as structured information. Such parameters are, Article Type, Secondary identifiers, Language, publication type parameter enables many special features. As these clinical girish can generate small sets of robust studies with considerable precision, since July 2005, the MEDLINE article indexing process extracts important identifiers from the article abstract and puts those in a field called Secondary Identifier. The secondary identifier field is to store numbers to various databases of molecular sequence data, gene expression or chemical compounds. For clinical trials, PubMed extracts trial IDs for the two largest trial registries, ClinicalTrials. gov and the International Standard Randomized Controlled Trial Number Register, a reference which is judged particularly relevant can be marked and related articles can be identified. If relevant, several studies can be selected and related articles to all of them can be generated using the Find related data option, the related articles are then listed in order of relatedness. To create these lists of related articles, PubMed compares words from the title and abstract of each citation, as well as the MeSH headings assigned, using a powerful word-weighted algorithm. The related articles function has been judged to be so precise that some researchers suggest it can be used instead of a full search, a strong feature of PubMed is its ability to automatically link to MeSH terms and subheadings. Examples would be, bad breath links to halitosis, heart attack to myocardial infarction, where appropriate, these MeSH terms are automatically expanded, that is, include more specific terms. Terms like nursing are automatically linked to Nursing or Nursing and this important feature makes PubMed searches automatically more sensitive and avoids false-negative hits by compensating for the diversity of medical terminology. The My NCBI area can be accessed from any computer with web-access, an earlier version of My NCBI was called PubMed Cubby

5.
National Center for Biotechnology Information
–
The National Center for Biotechnology Information is part of the United States National Library of Medicine, a branch of the National Institutes of Health. The NCBI is located in Bethesda, Maryland and was founded in 1988 through legislation sponsored by Senator Claude Pepper, the NCBI houses a series of databases relevant to biotechnology and biomedicine and is an important resource for bioinformatics tools and services. Major databases include GenBank for DNA sequences and PubMed, a database for the biomedical literature. Other databases include the NCBI Epigenomics database, all these databases are available online through the Entrez search engine. NCBI is directed by David Lipman, one of the authors of the BLAST sequence alignment program. He also leads a research program, including groups led by Stephen Altschul, David Landsman, Eugene Koonin, John Wilbur, Teresa Przytycka. NCBI is listed in the Registry of Research Data Repositories re3data. org, NCBI has had responsibility for making available the GenBank DNA sequence database since 1992. GenBank coordinates with individual laboratories and other databases such as those of the European Molecular Biology Laboratory. Since 1992, NCBI has grown to other databases in addition to GenBank. The NCBI assigns a unique identifier to each species of organism, the NCBI has software tools that are available by WWW browsing or by FTP. For example, BLAST is a sequence similarity searching program, BLAST can do sequence comparisons against the GenBank DNA database in less than 15 seconds. RAG2/IL2RG The NCBI Bookshelf is a collection of freely accessible, downloadable, some of the books are online versions of previously published books, while others, such as Coffee Break, are written and edited by NCBI staff. BLAST is a used for calculating sequence similarity between biological sequences such as nucleotide sequences of DNA and amino acid sequences of proteins. BLAST is a tool for finding sequences similar to the query sequence within the same organism or in different organisms. It searches the query sequence on NCBI databases and servers and post the results back to the browser in chosen format. Input sequences to the BLAST are mostly in FASTA or Genbank format while output could be delivered in variety of such as HTML, XML formatting. HTML is the output format for NCBIs web-page. Entrez is both indexing and retrieval system having data from sources for biomedical research

6.
Enzyme
–
Enzymes /ˈɛnzaɪmz/ are macromolecular biological catalysts. Enzymes accelerate, or catalyze, chemical reactions, the molecules at the beginning of the process upon which enzymes may act are called substrates and the enzyme converts these into different molecules, called products. Almost all metabolic processes in the cell need enzymes in order to occur at rates fast enough to sustain life, the set of enzymes made in a cell determines which metabolic pathways occur in that cell. The study of enzymes is called enzymology, enzymes are known to catalyze more than 5,000 biochemical reaction types. Most enzymes are proteins, although a few are catalytic RNA molecules, enzymes specificity comes from their unique three-dimensional structures. Like all catalysts, enzymes increase the rate of a reaction by lowering its activation energy, some enzymes can make their conversion of substrate to product occur many millions of times faster. An extreme example is orotidine 5-phosphate decarboxylase, which allows a reaction that would take millions of years to occur in milliseconds. Chemically, enzymes are like any catalyst and are not consumed in chemical reactions, enzymes differ from most other catalysts by being much more specific. Enzyme activity can be affected by other molecules, inhibitors are molecules that decrease enzyme activity, many drugs and poisons are enzyme inhibitors. An enzymes activity decreases markedly outside its optimal temperature and pH, some enzymes are used commercially, for example, in the synthesis of antibiotics. French chemist Anselme Payen was the first to discover an enzyme, diastase and he wrote that alcoholic fermentation is an act correlated with the life and organization of the yeast cells, not with the death or putrefaction of the cells. In 1877, German physiologist Wilhelm Kühne first used the term enzyme, the word enzyme was used later to refer to nonliving substances such as pepsin, and the word ferment was used to refer to chemical activity produced by living organisms. Eduard Buchner submitted his first paper on the study of yeast extracts in 1897, in a series of experiments at the University of Berlin, he found that sugar was fermented by yeast extracts even when there were no living yeast cells in the mixture. He named the enzyme that brought about the fermentation of sucrose zymase, in 1907, he received the Nobel Prize in Chemistry for his discovery of cell-free fermentation. Following Buchners example, enzymes are usually named according to the reaction they carry out, the biochemical identity of enzymes was still unknown in the early 1900s. Sumner showed that the enzyme urease was a protein and crystallized it. These three scientists were awarded the 1946 Nobel Prize in Chemistry, the discovery that enzymes could be crystallized eventually allowed their structures to be solved by x-ray crystallography. This high-resolution structure of lysozyme marked the beginning of the field of structural biology, an enzymes name is often derived from its substrate or the chemical reaction it catalyzes, with the word ending in -ase

7.
Catalysis
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Catalysis is the increase in the rate of a chemical reaction due to the participation of an additional substance called a catalyst. In most cases, reactions occur faster with a catalyst because they require less activation energy, furthermore, since they are not consumed in the catalyzed reaction, catalysts can continue to act repeatedly. Often only tiny amounts are required in principle, in the presence of a catalyst, less free energy is required to reach the transition state, but the total free energy from reactants to products does not change. A catalyst may participate in multiple chemical transformations, the effect of a catalyst may vary due to the presence of other substances known as inhibitors or poisons or promoters. Catalyzed reactions have an activation energy than the corresponding uncatalyzed reaction, resulting in a higher reaction rate at the same temperature. However, the mechanics of catalysis is complex. Usually, the catalyst participates in this slowest step, and rates are limited by amount of catalyst, in heterogeneous catalysis, the diffusion of reagents to the surface and diffusion of products from the surface can be rate determining. A nanomaterial-based catalyst is an example of a heterogeneous catalyst, analogous events associated with substrate binding and product dissociation apply to homogeneous catalysts. Although catalysts are not consumed by the reaction itself, they may be inhibited, deactivated, in heterogeneous catalysis, typical secondary processes include coking where the catalyst becomes covered by polymeric side products. Additionally, heterogeneous catalysts can dissolve into the solution in a system or sublimate in a solid–gas system. The production of most industrially important chemicals involves catalysis, similarly, most biochemically significant processes are catalysed. Research into catalysis is a field in applied science and involves many areas of chemistry, notably organometallic chemistry. Catalysis is relevant to aspects of environmental science, e. g. the catalytic converter in automobiles. Many transition metals and transition metal complexes are used in catalysis as well, Catalysts called enzymes are important in biology. A catalyst works by providing a reaction pathway to the reaction product. The rate of the reaction is increased as this route has a lower activation energy than the reaction route not mediated by the catalyst. The disproportionation of hydrogen peroxide creates water and oxygen, as shown below,2 H2O2 →2 H2O + O2 This reaction is preferable in the sense that the reaction products are more stable than the starting material, though the uncatalysed reaction is slow. In fact, the decomposition of hydrogen peroxide is so slow that hydrogen peroxide solutions are commercially available and this reaction is strongly affected by catalysts such as manganese dioxide, or the enzyme peroxidase in organisms

8.
Chemical reaction
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A chemical reaction is a process that leads to the transformation of one set of chemical substances to another. Nuclear chemistry is a sub-discipline of chemistry that involves the reactions of unstable. The substance initially involved in a reaction are called reactants or reagents. Chemical reactions are characterized by a chemical change, and they yield one or more products. Reactions often consist of a sequence of individual sub-steps, the elementary reactions. Chemical reactions are described with chemical equations, which present the starting materials, end products. Chemical reactions happen at a characteristic reaction rate at a given temperature, typically, reaction rates increase with increasing temperature because there is more thermal energy available to reach the activation energy necessary for breaking bonds between atoms. Reactions may proceed in the forward or reverse direction until they go to completion or reach equilibrium, Reactions that proceed in the forward direction to approach equilibrium are often described as spontaneous, requiring no input of free energy to go forward. Non-spontaneous reactions require input of energy to go forward. Different chemical reactions are used in combinations during chemical synthesis in order to obtain a desired product, in biochemistry, a consecutive series of chemical reactions form metabolic pathways. These reactions are catalyzed by protein enzymes. Chemical reactions such as combustion in fire, fermentation and the reduction of ores to metals were known since antiquity, in the Middle Ages, chemical transformations were studied by Alchemists. They attempted, in particular, to lead into gold, for which purpose they used reactions of lead. The process involved heating of sulfate and nitrate minerals such as sulfate, alum. In the 17th century, Johann Rudolph Glauber produced hydrochloric acid and sodium sulfate by reacting sulfuric acid, further optimization of sulfuric acid technology resulted in the contact process in the 1880s, and the Haber process was developed in 1909–1910 for ammonia synthesis. From the 16th century, researchers including Jan Baptist van Helmont, Robert Boyle, the phlogiston theory was proposed in 1667 by Johann Joachim Becher. It postulated the existence of an element called phlogiston, which was contained within combustible bodies. This proved to be false in 1785 by Antoine Lavoisier who found the explanation of the combustion as reaction with oxygen from the air

9.
Pyrophosphate
–
In chemistry, a pyrophosphate is a phosphorus oxyanion. Compounds such as salts and esters are called pyrophosphates. The group is also called diphosphate or dipolyphosphate, although this should not be confused with phosphates, as a food additive, diphosphates are known as E450. A number of hydrogen pyrophosphates also exist, such as Na2H2P2O7, pyrophosphates were originally prepared by heating phosphates. They generally exhibit the highest solubilities among the phosphates, moreover, pyrophosphate is the first member of an entire series of polyphosphates. The term pyrophosphate is also the name of esters formed by the condensation of a phosphorylated biological compound with inorganic phosphate and this bond is also referred to as a high-energy phosphate bond. The synthesis of tetraethyl pyrophosphate was first described in 1854 by Philippe de Clermont at a meeting of the French Academy of Sciences, pyrophosphates are very important in biochemistry. The anion P2O74− is abbreviated PPi and is formed by the hydrolysis of ATP into AMP in cells, ATP → AMP + PPi For example, when a nucleotide is incorporated into a growing DNA or RNA strand by a polymerase, pyrophosphate is released. The pyrophosphate anion has the structure P2O74−, and is an anhydride of phosphate. This hydrolysis to inorganic phosphate effectively renders the cleavage of ATP to AMP and PPi irreversible, PPi occurs in synovial fluid, blood plasma, and urine at levels sufficient to block calcification and may be a natural inhibitor of hydroxyapatite formation in extracellular fluid. Cells may channel intracellular PPi into ECF, ANK is a nonenzymatic plasma-membrane PPi channel that supports extracellular PPi levels. Defective function of the membrane PPi channel ANK is associated with low extracellular PPi, ectonucleotide pyrophosphatase/phosphodiesterase may function to raise extracellular PPi. AMP + ATP →2 ADP2 ADP +2 Pi →2 ATP The plasma concentration of inorganic pyrophosphate has a range of 0. 58-3.78 µM. Various diphosphates are used as emulsifiers, stabilisers, acidity regulators, raising agents, sequestrants, schröder HC, Kurz L, Muller WE, Lorenz B. Pyrophosphates at the US National Library of Medicine Medical Subject Headings

10.
Melon
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A melon is any of various plants of the family Cucurbitaceae with sweet edible, fleshy fruit. The word melon can refer to either the plant or specifically to the fruit, botanically, a melon is a kind of berry, specifically a pepo. The word melon derives from Latin melopepo, which is the latinization of the Greek μηλοπέπων, meaning melon, itself a compound of μῆλον, apple and πέπων, many different cultivars have been produced, particularly of muskmelons. Melons originated in Africa and southwest Asia, but they began to appear in Europe toward the end of the Roman Empire. Melons were among the earliest plants to be domesticated in both the Old and New Worlds, early European settlers in the New World are recorded as growing honeydew and casaba melons as early as the 1600s. Organizations like Native Seeds/SEARCH have made an effort to collect and preserve these, winter melon is the only member of the genus Benincasa. The mature winter melon is a vegetable that is widely used in Asia and India. The immature melons are used as a culinary fruit, egusi is a wild melon, similar in appearance to the watermelon. The flesh is inedible, but the seeds are a food source in Africa. Other species that have the same role, and that are also called egusi include Cucumeropsis mannii. Watermelon originated in Africa, where evidence indicates that it has been cultivated for over 4,000 years and it is a popular summer fruit in all parts of the world. Melons in genus Cucumis are culinary fruits, and include the majority of culinary melons, all but a handful of culinary melon varieties belong to the species Cucumis melo L. Horned melon, a traditional food plant in Africa with distinctive spikes. Now grown in California, Chile, Australia and New Zealand as well, muskmelon C. melo cantalupensis, with skin that is rough and warty, not netted. The European cantaloupe, with lightly ribbed, pale skin, was domesticated in the 18th century, in Cantalupo in Sabina, Italy. Varieties include the French Charentais and the Burpee Seeds hybrid Netted Gem, the Yubari King is a highly prized Japanese cantaloupe cultivar. The Persian melon resemble a large cantaloupe with a darker green rind, C. melo inodorus, casabas, honeydew, and Asian melons Argos, a large, oblong, with orange wrinkled skin, orange flesh, strong aroma. A characteristic is its pointed ends, growing in some areas of Greece, from which it was named. Canary melon, a large, bright-yellow melon with a green to white inner flesh

11.
Muskmelon
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Muskmelon is a species of melon that has been developed into many cultivated varieties. These include smooth-skinned varieties such as honeydew, Crenshaw, and casaba, the Armenian cucumber is also a variety of muskmelon, but its shape, taste, and culinary uses more closely resemble those of a cucumber. The large number of cultivars in this species approaches that found in wild cabbage and it is a fruit of a type called pepo. Muskmelon is native to Iran, Anatolia and the Caucasus, with a center including northwest India. They do not cross with watermelon, cucumber, pumpkin, or squash, the genome of Cucumis melo L. was first sequenced in 2012. Per 100 gram serving, cantaloupe melons provide 34 calories and are an excellent source of vitamin A and vitamin C, Melons are 90% water and 9% carbohydrates, with less than 1% each of protein and fat. In addition to their consumption when fresh, melons are sometimes dried, other varieties are cooked, or grown for their seeds, which are processed to produce melon oil. Still other varieties are only for their pleasant fragrance. The Japanese liqueur, Midori, is flavored with muskmelon, seeds Handbook, Biology, Production, Processing, and Storage, Vol.103. A portable dictionary of the higher plants, food and feed crops of the United States. Interregional Research Project IR-4, IR Bul, Cucumis melo L. – Purdue University, Center for New Crops & Plant Products. Sorting Cucumis names – Multilingual multiscript plant name database Cooks Thesaurus, Melons – Varietal names and pictures

12.
PubMed Identifier
–
PubMed is a free search engine accessing primarily the MEDLINE database of references and abstracts on life sciences and biomedical topics. The United States National Library of Medicine at the National Institutes of Health maintains the database as part of the Entrez system of information retrieval, from 1971 to 1997, MEDLINE online access to the MEDLARS Online computerized database primarily had been through institutional facilities, such as university libraries. PubMed, first released in January 1996, ushered in the era of private, free, home-, the PubMed system was offered free to the public in June 1997, when MEDLINE searches via the Web were demonstrated, in a ceremony, by Vice President Al Gore. Information about the journals indexed in MEDLINE, and available through PubMed, is found in the NLM Catalog. As of 5 January 2017, PubMed has more than 26.8 million records going back to 1966, selectively to the year 1865, and very selectively to 1809, about 500,000 new records are added each year. As of the date,13.1 million of PubMeds records are listed with their abstracts. In 2016, NLM changed the system so that publishers will be able to directly correct typos. Simple searches on PubMed can be carried out by entering key aspects of a subject into PubMeds search window, when a journal article is indexed, numerous article parameters are extracted and stored as structured information. Such parameters are, Article Type, Secondary identifiers, Language, publication type parameter enables many special features. As these clinical girish can generate small sets of robust studies with considerable precision, since July 2005, the MEDLINE article indexing process extracts important identifiers from the article abstract and puts those in a field called Secondary Identifier. The secondary identifier field is to store numbers to various databases of molecular sequence data, gene expression or chemical compounds. For clinical trials, PubMed extracts trial IDs for the two largest trial registries, ClinicalTrials. gov and the International Standard Randomized Controlled Trial Number Register, a reference which is judged particularly relevant can be marked and related articles can be identified. If relevant, several studies can be selected and related articles to all of them can be generated using the Find related data option, the related articles are then listed in order of relatedness. To create these lists of related articles, PubMed compares words from the title and abstract of each citation, as well as the MeSH headings assigned, using a powerful word-weighted algorithm. The related articles function has been judged to be so precise that some researchers suggest it can be used instead of a full search, a strong feature of PubMed is its ability to automatically link to MeSH terms and subheadings. Examples would be, bad breath links to halitosis, heart attack to myocardial infarction, where appropriate, these MeSH terms are automatically expanded, that is, include more specific terms. Terms like nursing are automatically linked to Nursing or Nursing and this important feature makes PubMed searches automatically more sensitive and avoids false-negative hits by compensating for the diversity of medical terminology. The My NCBI area can be accessed from any computer with web-access, an earlier version of My NCBI was called PubMed Cubby

13.
Carbonic anhydrase
–
The active site of most carbonic anhydrases contains a zinc ion, they are therefore classified as metalloenzymes. Typical catalytic rates of the different forms of this enzyme ranging between 104 and 106 reactions per second, the reverse reaction is relatively slow in the absence of a catalyst. H C O3 − + H + → H2 C O3 → C O2 + H2 O A zinc prosthetic group in the enzyme is coordinated in three positions by histidine side-chains, the fourth coordination position is occupied by water. This causes polarisation of the bond, making the oxygen slightly more positive. A fourth histidine is placed close to the substrate of water and accepts a proton and this leaves a hydroxide attached to the zinc. The active site contains a specificity pocket for carbon dioxide. This allows the electron-rich hydroxide to attack the carbon dioxide, forming bicarbonate, there are at least five distinct CA families. These families have no significant amino acid sequence similarity and in most cases are thought to be an example of convergent evolution, the α-CAs are found in humans. Vertebrates, algae and some bacteria have this family of CAs, most prokaryotic and plant chloroplast CAs belong to the beta family. Two signature patterns for this family have been identified, C--D-S-R--x- --A--x--x--x-G-H-x-C-G The gamma class of CAs come from methanogens, the delta class of CAs has been described in diatoms. The distinction of class of CA has recently come into question. The zeta class of CAs occurs exclusively in bacteria in a few chemolithotrophs, recent 3-dimensional analyses suggest that ζ-CA bears some structural resemblance to β-CA, particularly near the metal ion site. Thus, the two forms may be related, even though the underlying amino acid sequence has since diverged considerably. The eta family of CAs was recently found in organisms of the genus Plasmodium and these are a group of enzymes previously thought to belong to the alpha family of CAs, however it has been demonstrated that η-CAs have unique features, such as their metal ion coordination pattern. Several forms of carbonic anhydrase occur in nature, in the best-studied α-carbonic anhydrase form present in animals, the zinc ion is coordinated by the imidazole rings of 3 histidine residues, His94, His96, and His119. There are at least 14 different isoforms in mammals, in plants, carbonic anhydrase helps raise the concentration of CO2 within the chloroplast in order to increase the carboxylation rate of the enzyme RuBisCO. This is the reaction that integrates CO2 into organic carbon sugars during photosynthesis, marine diatoms have been found to express a new form of ζ carbonic anhydrase. T. weissflogii, a species of phytoplankton common to marine ecosystems, was found to contain carbonic anhydrase with a cadmium ion in place of zinc

14.
Fumarase
–
Fumarase is an enzyme that catalyzes the reversible hydration/dehydration of fumarate to malate. Fumarase comes in two forms, mitochondrial and cytosolic, the mitochondrial isoenzyme is involved in the Krebs Cycle, and the cytosolic isoenzyme is involved in the metabolism of amino acids and fumarate. This enzyme participates in 2 metabolic pathways, citric acid cycle, reductive citric acid cycle, mutations in this gene have been associated with the development of leiomyomas in the skin and uterus in combination with renal cell carcinoma. This enzyme belongs to the family of lyases, specifically the hydro-lyases, the systematic name of this enzyme class is -malate hydro-lyase. Other names in use include, fumarase L-malate hydro-lyase -malate hydro-lyase The FH gene is localized to the chromosomal position 1q42. 3-q43. The FH gene contains 10 exons, crystal structures of fumarase C from Escherichia coli have been observed to have two occupied dicarboxylate binding sites. These are known as the site and the B site. The active site and B site are identified as having areas unoccupied by a bound ligand. This so-called ‘free’ crystal structure demonstrates conservation of the active-site water, similar orientation has been discovered in other fumarase C crystal structures. Crystallographic research on the B site of the enzyme has observed that there is a shift on His129 and this information suggests that water is a permanent component of the active site. It also suggests that the use of an imidazole-imidazolium conversion controls access to the allosteric B site, figure 2 depicts the fumarase reaction mechanism. Two acid-base groups catalyze proton transfer, and the state of these groups is in part defined by two forms of the enzyme E1 and E2. In E1, the groups exist in an internally neutralized A-H/B, state, while in E2, E1 binds fumarate and facilitates its transformation into malate, and E2 binds malate and facilitates its transformation into fumarate. The two forms must undergo isomerization with each catalytic turnover, despite its biological significance, the reaction mechanism of fumarase is not completely understood. This led to the conclusion that in the formation of S-Malate from fumarate E1 elimination, protonation of fumarate to the carbocation was followed by the additional of a group from H2O. However, more recent trials have provided evidence that the mechanism actually takes place through an acid-base catalyzed elimination by means of a carbanionic intermediate E1CB elimination. The function of fumarase in the citric acid cycle is to facilitate a transition step in the production of energy in the form of NADH, in the cytosol the enzyme functions to metabolize fumarate, which is a byproduct of the urea cycle as well as amino acid catabolism. Studies have revealed that the site is composed of amino acid residues from three of the four subunits within the tetrameric enzyme

15.
Aconitase
–
Aconitase is an enzyme that catalyses the stereo-specific isomerization of citrate to isocitrate via cis-aconitate in the tricarboxylic acid cycle, a non-redox-active process. Aconitase, displayed in the structures in the margin of this page, has two slightly different structures, depending on whether it is activated or inactivated. In the inactive form, its structure is divided into four domains, the Fe-S cluster and a SO42− anion also reside in the active site. When the enzyme is activated, it gains an additional iron atom, however, the structure of the rest of the enzyme is nearly unchanged, the conserved atoms between the two forms are in essentially the same positions, up to a difference of 0.1 angstroms. In contrast with the majority of proteins that function as electron carriers. Aconitase has an active 2+ cluster, which may convert to an inactive + form, three cysteine residues have been shown to be ligands of the centre. In the active state, the iron ion of the cluster is not coordinated by Cys. The iron-responsive element-binding protein and 3-isopropylmalate dehydratase, an enzyme catalysing the second step in the biosynthesis of leucine, are known aconitase homologues, iron regulatory elements constitute a family of 28-nucleotide, non-coding, stem-loop structures that regulate iron storage, heme synthesis and iron uptake. They also participate in ribosome binding and control the mRNA turnover, the specific regulator protein, the IRE-BP, binds to IREs in both 5 and 3 regions, but only to RNA in the apo form, without the Fe-S cluster. Mutant IRE-BPs, in any or all of the three Cys residues involved in Fe-S formation are replaced by serine, have no aconitase activity. Aconitase is inhibited by fluoroacetate, therefore fluoroacetate is poisonous, fluoroacetate, in the citric acid cycle, can innocently enter as fluorocitrate. However, aconitase cannot bind this substrate and thus the citric acid cycle is halted, the iron sulfur cluster is highly sensitive to oxidation by superoxide. The catalytic residues involved are His-101 and Ser-642, at this point, the intermediate is rotated 180°. This rotation is referred to as a flip, because of this flip, the intermediate is said to move from a citrate mode to a isocitrate mode. How exactly this flip occurs is debatable, one theory is that, in the rate-limiting step of the mechanism, the cis-aconitate is released from the enzyme, then reattached in the isocitrate mode to complete the reaction. This rate-liming step ensures that the right stereochemistry, specifically, is formed in the final product, another hypothesis is that cis-aconitate stays bound to the enzyme while it flips from the citrate to the isocitrate mode. In either case, flipping cis-aconitate allows the dehydration and hydration steps to occur on faces of the intermediate. Aconitase catalyzes trans elimination/addition of water, and the guarantees that the correct stereochemistry is formed in the product

16.
Enolase
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The systematic name of this enzyme is 2-phospho-D-glycerate hydro-lyase. The reaction is reversible, depending on environmental concentrations of substrates, the optimum pH for the human enzyme is 6.5. Enolase is present in all tissues and organisms capable of glycolysis or fermentation, the enzyme was discovered by Lohmann and Meyerhof in 1934, and has since been isolated from a variety of sources including human muscle and erythrocytes. In humans, deficiency of ENO1 is linked to hereditary haemolytic anemia while ENO3 deficiency is linked to glycogen storage disease XIII. In humans there are three subunits of enolase, α, β, and γ, each encoded by a gene that can combine to form five different isoenzymes, αα, αβ, αγ, ββ. It is present at some level in all human cells. Also known as enolase 1 ββ or muscle specific enolase and this enzyme is largely restricted to muscle where it is present at very high levels in muscle γγ or neuron-specific enolase. Expressed at very high levels in neurons and neural tissues, where it can account for as much as 3% of total soluble protein and it is expressed at much lower levels in most mammalian cells. When present in the cell, different isozymes readily form heterodimers. Enolase is a member of the enolase superfamily. It has a weight of 82, 000-100,000 Daltons depending on the isoform. In human alpha enolase, the two subunits are antiparallel in orientation so that Glu20 of one forms an ionic bond with Arg414 of the other subunit. Each subunit has two distinct domains, the smaller N-terminal domain consists of three α-helices and four β-sheets. The enzyme’s compact, globular structure results from significant hydrophobic interactions between two domains. Enolase is a highly conserved enzyme with five active-site residues being especially important for activity, when compared to wild-type enolase, a mutant enolase that differs at either the Glu168, Glu211, Lys345, or Lys396 residue has an activity level that is cut by a factor of 105. Also, changes affecting His159 leave the mutant with only 0. 01% of its catalytic activity, an integral part of enolase are two Mg2+ cofactors in the active site, which serve to stabilize negative charges in the substrate. Recently, moonlighting functions of several enolases, such as interaction with plasminogen, have brought interest to the enzymes catalytic loops, using isotopic probes, the overall mechanism for converting 2-PG to PEP is proposed to be an E1cb elimination reaction involving a carbanion intermediate. The following detailed mechanism is based on studies of crystal structure, when the substrate, 2-phosphoglycerate, binds to α-enolase, its carboxyl group coordinates with two magnesium ion cofactors in the active site

17.
Alpha-enolase
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Enolase 1, more commonly known as alpha-enolase, is a glycolytic enzyme expressed in most tissues, one of the isozymes of enolase. Each isoenzyme is a composed of 2 alpha,2 gamma, or 2 beta subunits. Alpha-enolase, in addition, functions as a structural protein in the monomeric form. Alternative splicing of this results in a shorter isoform that has been shown to bind to the c-myc promoter. Several pseudogenes have been identified, including one on the arm of chromosome 1. Alpha-enolase has also identified as an autoantigen in Hashimoto encephalopathy. ENO1 is one of three isoforms, the other two being ENO2 and ENO3. Each isoform is a subunit that can hetero- or homodimerize to form αα, αβ, αγ, ββ. The ENO1 gene spans 18 kb and lacks a TATA box while possessing multiple transcription start sites, a hypoxia-responsive element can be found in the ENO1 promoter and allows the enzyme to function in aerobic glycolysis and contribute to the Warburg effect in tumor cells. The mRNA transcript of the ENO1 gene can be translated into a cytoplasmic protein, with a molecular weight of 48 kDa, or a nuclear protein. The nuclear form was identified as Myc-binding protein-1, which downregulates the protein level of the c-myc protooncogene. A start codon at codon 97 of ENO1 and a Kozak consensus sequence were found preceding the 3 region of ENO1 encoding the MBP1 protein, in addition, the N-terminal region of the MBP1 protein it critical to DNA binding and, thus, its inhibitory function. As an enolase, ENO1 is a glycolytic enzyme the catalyzes the conversion of 2-phosphoglycerate to phosphoenolpyruvate and this isozyme is ubiquitously expressed in adult human tissues, including liver, brain, kidney, and spleen. Within cells, ENO1 predominantly localizes to the cytoplasm, though an alternatively translated form is localizes to the nucleus, in many of these tumors, ENO1 promoted cell proliferation by regulating the PI3K/AKT signaling pathway and induced tumorigenesis by activating plasminogen. Moreover, ENO1 is expressed on the cell surface during pathological conditions such as inflammation, autoimmunity. Its role as a receptor leads to extracellular matrix degradation. Due to its expression, targeting surface ENO1 enables selective targeting of tumor cells while leaving the ENO1 inside normal cells functional. Considering these factors, ENO1 holds great potential to serve as a therapeutic target for treating many types of tumors in patients

18.
Enolase 2
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Gamma-enolase, also known as enolase 2 or neuron specific enolase, is an enzyme that in humans is encoded by the ENO2 gene. Gamma-enolase is one of the three enolase isoenzymes found in mammals and this isoenzyme, a homodimer, is found in mature neurons and cells of neuronal origin. A switch from alpha enolase to gamma enolase occurs in tissue during development in rats. Click on genes, proteins and metabolites below to link to respective articles, detection of NSE with antibodies can be used to identify neuronal cells and cells with neuroendocrine differentiation. NSE is produced by small cell carcinomas which are neuroendocrine in origin, NSE is therefore a useful tumor marker for lung cancer patients

19.
Enoyl-CoA hydratase
–
Enoyl-CoA hydratase is an enzyme that hydrates the double bond between the second and third carbons on acyl-CoA. This enzyme, also known as crotonase, is essential to metabolizing fatty acids to produce both acetyl CoA and energy, note the crystal structure at right of enoyl-coa hydratase from a rat. The crystal structure shows a hexamer formation, which leads to the efficiency of this protein and this enzyme has been discovered to be highly efficient, and allows our bodies to metabolize fatty acids into energy very quickly. In fact this enzyme is so efficient that the rate is equivalent to that of diffusion-controlled reactions, enoyl-CoA hydratase catalyzes the second step in the breakdown of fatty acids or the second step of β-oxidation in fatty acid metabolism shown below. Fatty acid metabolism is how our bodies turn fats or lipids into energy, when fats come into our bodies, they are generally in the form of triacyl-glycerols. These must be broken down in order for the fats to pass into our bodies, when that happens, three fatty acids are released. In fatty acid metabolism, fatty acids are changed into fatty acyl-CoA, to do this, the carboxylate which occupies one end of the fatty acid is changed into a thioester by substituting coenzyme A for the hydroxyl group. Next the fatty acyl-CoA is oxidized and broken down into an acetyl-CoA molecule, the acetyl CoA is then sent to the citric acid cycle while the remaining acyl-CoA is broken down further into acetyl-CoAs. The complete breakdown of a fatty acid not only generates acetyl-CoA molecules and this NADH goes on to be converted into ATP which can be used in other reactions. Enoyl-CoA hydratase is used in β-oxidation to add a hydroxyl group, the enzyme functions by providing two glutamate residues as catalytic acid and base. The two amino acids hold a molecule in place, allowing it to attack in a syn addition to an α-β unsaturated acyl-CoA at the β-carbon. The α-carbon then grabs another proton, which completes the formation of the beta-hydroxy acyl-CoA and it is also known from experimental data that no other sources of protons reside in the active site. This means that the proton which the α-carbon grabs is from the water that just attacked the β-carbon, what this implies is that the hydroxyl group and the proton from water are both added from the same side of the double bond, a syn addition. This allows the enzyme to make an S stereoisomer from 2-trans-enoyl-CoA and this is made possible by the two glutamate residues which hold the water in position directly adjacent to the α-β unsaturated double bond, as seen in figure 1. This configuration requires that the site for this enzyme is extremely rigid. The data for a mechanism for this reaction is not conclusive as to whether this reaction is concerted or occurs in consecutive steps, if occurring in consecutive steps, the intermediate is identical to that which would be generated from an E1cb elimination reaction. The enzyme is similar to fumarase. It is classified as EC4.2.1.17, enoyl-CoA Hydratase at the US National Library of Medicine Medical Subject Headings

20.
Tryptophan synthase
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Tryptophan synthase or tryptophan synthetase is an enzyme that catalyzes the final two steps in the biosynthesis of tryptophan. It is commonly found in Eubacteria, Archaebacteria, Protista, Fungi, however, it is absent from Animalia. It is typically found as an α2β2 tetramer, the α subunits catalyze the reversible formation of indole and glyceraldehyde-3-phosphate from indole-3-glycerol phosphate. The β subunits catalyze the condensation of indole and serine to form tryptophan in a pyridoxal phosphate dependent reaction. Each α active site is connected to a β active site by a 25 angstrom long hydrophobic channel contained within the enzyme and this facilitates the diffusion of indole formed at α active sites directly to β active sites in a process known as substrate channeling. The active sites of tryptophan synthase are allosterically coupled, subunits, Tryptophan synthase typically exists as an α-ββ-α complex. The α and β subunits have molecular masses of 27 and 43 kDa respectively, the α subunit has a TIM barrel conformation. The β subunit has a fold type II conformation and a site adjacent to the active site for monovalent cations. Their assembly into a complex leads to changes in both subunits resulting in reciprocal activation. There are two mechanisms for intersubunit communication. First, the COMM domain of the β-subunit and the α-loop2 of the α-subunit interact, additionally, there are interactions between the αGly181 and βSer178 residues. The active sites are regulated allosterically and undergo transitions between open, inactive, and closed, active, states, indole-3-glycerol binding site, See image 1. Indole and serine binding site, See image 1, hydrophobic channel, The α and β active sites are separated by a 25 angstrom long hydrophobic channel contained within the enzyme allowing for the diffusion of indole. If the channel did not exist, the indole formed at an α active site would quickly diffuse away and be lost to the cell as it is hydrophobic, as such, the channel is essential for enzyme complex function. α subunit reaction, The α subunit catalyzes the formation of indole, the αGlu49 and αAsp60 are thought to be directly involved in the catalysis as shown. The rate limiting step is the isomerization of IGP, β subunit reaction, The β subunit catalyzes the β-replacement reaction in which indole and serine condense to form tryptophan in a PLP dependent reaction. The βLys87, βGlu109, and βSer377 are thought to be involved in the catalysis as shown. Again, the mechanism has not been conclusively determined

21.
Cystathionine beta synthase
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Cystathionine-β-synthase, also known as CBS, is an enzyme that in humans is encoded by the CBS gene. This enzyme belongs to the family of lyases, to be specific, the hydro-lyases, CBS is a multidomain enzyme composed of an N-terminal enzymatic domain and two CBS domains. The CBS gene is the most common locus for mutations associated with homocystinuria, the systematic name of this enzyme class is L-serine hydro-lyase. Other names in use include, beta-thionase, cysteine synthase, L-serine hydro-lyase, methylcysteine synthase, serine sulfhydrase. Methylcysteine synthase was assigned the EC number EC4.2.1.23 in 1961, a side-reaction of CBS caused this. The EC number EC4.2.1.23 was deleted in 1972, the human enzyme cystathionine β-synthase is a tetramer and comprises 551 amino acids with a subunit molecular weight of 61 kDa. It displays a modular organization of three modules with the N-terminal heme domain followed by a core contains the PLP cofactor. The cofactor is deep in the domain and is linked by a Schiff base. A Schiff base is a group containing a C=N bond with the nitrogen atom connected to an aryl or alkyl group. The heme domain is composed of 70 amino acids and it appears that the heme only exists in mammalian CBS and is absent in yeast and protozoan CBS. At the C-terminus, the domain of CBS contains a tandem repeat of two CBS domains of β-α-β-β-α, a secondary structure motif found in other proteins. CBS has a C-terminal inhibitory domain, the C-terminal domain of cystathionine β-synthase regulates its activity via both intrasteric and allosteric effects and is important for maintaining the tetrameric state of the protein. This inhibition is alleviated by binding of the effector, adoMet, or by deletion of the regulatory domain, however. Mutations in this domain are correlated with hereditary diseases, the heme domain contains an N-terminal loop that binds heme and provides the axial ligands C52 and H65. The presence of protoporphyrin IX in CBS is a unique PLP-dependent enzyme and is found in the mammalian CBS. D. melanogaster and D. discoides have truncated N-terminal extensions, however, the Anopheles gambiae sequence has a longer N-terminal extension than the human enzyme and contains the conserved histidine and cysteine heme ligand residues like the human heme. The PLP is an internal aldimine and forms a Schiff base with K119 in the active site, between the catalytic and regulatory domains exists a hypersensitive site that causes proteolytic cleavage and produces a truncated dimeric enzyme that is more active than the original enzyme. Both truncated enzyme and the found in yeast are not regulated by adoMet

22.
Porphobilinogen synthase
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Porphobilinogen synthase synthesizes porphobilinogen through the asymmetric condensation of two molecules of aminolevulinic acid. All natural tetrapyrroles, including hemes, chlorophylls and vitamin B12, porphobilinogen synthase is the prototype morpheein. The * represents a reorientation between two domains of each subunit that occurs in the state because it is sterically forbidden in the larger multimers. PBGS is encoded by a gene and each PBGS multimer is composed of multiple copies of the same protein. Each PBGS subunit consists of a ~300 residue αβ-barrel domain, which houses the enzymes active site in its center, allosteric regulation of PBGS can be described in terms of the orientation of the αβ-barrel domain with respect to the N-terminal arm domain. Each N-terminal arm has up to two interactions with subunits in a PBGS multimer. One of these interactions helps to stabilize a conformation of the active site lid. The other interaction restricts solvent access from the end of the αβ-barrel. As a nearly universal enzyme with a conserved active site. To the contrary, allosteric sites can be much more variable than active sites. Phylogenetic variation in PBGS allostery leads to the framing of discussion of PBGS allosteric regulation in terms of intrinsic and extrinsic factors, the allosteric magnesium ion lies at the highly hydrated interface of two pro-octamer dimers. It appears to be easily dissociable, and it has shown that hexamers accumulate when magnesium is removed in vitro. Inspection of the PBGS 6mer* reveals a surface cavity that is not present in the 8mer, small molecule binding to this phylogenetically variable cavity has been proposed to stabilize 6mer* of the targeted PBGS and consequently inhibit activity. Such allosteric regulators are known as morphlocks because they lock PBGS in a specific morpheein form, a deficiency of porphobilinogen synthase is usually acquired and can be caused by heavy metal poisoning, especially lead poisoning, as the enzyme is very susceptible to inhibition by heavy metals. Hereditary insufficiency of porphobilinogen synthase is called porphobilinogen synthase deficiency poprhyria and it is an extremely rare cause of porphyria, with less than 10 cases ever reported. All disease associated protein variants favor hexamer formation relative to the wild type human enzyme, lead poisoning works on the cellular level by binding to this enzyme, rendering it useless. Delta-Aminolevulinic Acid Dehydratase at the US National Library of Medicine Medical Subject Headings http, //www. omim. org/entry/125270. search=pbgs&highlight=pbgs

23.
Urocanase
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Urocanase is the enzyme that catalyzes the second step in the degradation of histidine, the hydration of urocanate into imidazolonepropionate. Urocanase is coded for by the UROC1 gene, located on the 3rd chromosome in humans, the protein itself is composed of 676 amino acids which then fold, producing the final product which has 2 identical subunits, making the enzyme a homodimer. To catalyze the hydrolysis of urocanate in the pathway of L-histidine the enzyme utilizes its two NAD+ groups. The NAD+ groups act as electrophiles, attaching to the top carbon of the urocanate which leads to sigmatropic rearrangement of the urocanate molecule and this rearrangement allows for the addition of a water molecule, converting the urocanate into 4, 5-dihydro-4-oxo-5-imidazolepropanoate. Urocanate + H2O ⇌4, 5-dihydro-4-oxo-5-imidazolepropanoate Inherited deficiency of urocanase leads to elevated levels of acid in the urine. Urocanase is found in bacteria, in the liver of many vertebrates and has also been found in the plant Trifolium repens. Urocanase is a protein of about 60 Kd, it binds tightly to NAD+, a conserved cysteine has been found to be important for the catalytic mechanism and could be involved in the binding of the NAD+. Urocanate Hydratase at the US National Library of Medicine Medical Subject Headings EC4.2.1.49

24.
Uroporphyrinogen III synthase
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Uroporphyrinogen III synthase EC4.2.1.75 is an enzyme involved in the metabolism of the cyclic tetrapyrrole compound porphyrin. It is involved in the conversion of hydroxymethyl bilane into uroporphyrinogen III, the enzyme folds into two alpha/beta domains connected by a beta-ladder, the active site being located between the two domains. A deficiency is associated with Gunthers disease, also known as congenital erythropoietic porphyria and this is an autosomal recessive inborn error of metabolism that results from the markedly deficient activity of uroporphyrinogen III synthase. Uroporphyrinogen III synthase at the US National Library of Medicine Medical Subject Headings This article incorporates text from the public domain Pfam and InterPro IPR003754

25.
Nitrile hydratase
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Nitrile hydratase is one of the rare enzyme types that use cobalt in a non-corrinoid manner. The mechanism by which the cobalt is transported to NHase without causing toxicity is unclear, although a cobalt permease has been identified, which transports cobalt across the cell membrane. The identity of the metal in the site of a nitrile hydratase can be predicted by analysis of the sequence data of the alpha subunit in the region where the metal is bound. The presence of the amino acid sequence VCTLC indicates a Co-centred NHase, a sequence in genome of the choanoflagellate Monosiga brevicollis was suggested to encode for a nitrile hydratase. The M. brevicollis gene consisted of both the alpha and beta subunits fused into a single gene, NHases have been efficiently used for the industrial production of acrylamide from acrylonitrile and for removal of nitriles from wastewater. Photosensitive NHases intrinsically possess nitric oxide bound to the iron centre, NHases are composed of two types of subunits, α and β, which are not related in amino acid sequence. NHases exist as αβ dimers or α2β2 tetramers and bind one metal atom per αβ unit, the 3-D structures of a number of NHases have been determined. The α subunit consists of a long extended N-terminal arm, containing two α-helices, and a C-terminal domain with an unusual four-layered structure, an assembly pathway for nitrile hydratase was first proposed when gel filtration experiments found that the complex exists in both αβ and α2β2 forms. In vitro experiments using mass spectrometry further revealed that the α and β subunits first assemble to form the αβ dimer, the dimers can then subsequently interact to form a tetramer. The metal centre is located in the cavity at the interface between two subunits. All protein ligands to the atom are provided by the α subunit. The protein ligands to the iron are the sidechains of the three cysteine residues and two mainchain amide nitrogens, the metal ion is octahedrally coordinated, with the protein ligands at the five vertices of an octahedron. The sixth position, accessible to the active site cleft, is occupied either by NO or by a solvent-exchangeable ligand, the two Cys residues coordinated to the metal are post-translationally modified to Cys-sulfinic and -sulfenic acids. Quantum chemical studies predicted that the Cys-SOH residue might play a role as either a base or as a nucleophile, subsequently, the functional role of the SOH center as nucleophile has obtained experimental support

26.
Active site
–
In biology, the active site is the region of an enzyme where substrate molecules bind and undergo a chemical reaction. The active site consists of residues that form bonds with the substrate. The active site is usually a groove or pocket of the enzyme which can be located in a tunnel within the enzyme. An active site can catalyse a reaction repeatedly as its residues are not altered at the end of the reaction, usually, an enzyme molecule has only one active site, and the active site fits with one specific type of substrate. An active site contains a site that binds the substrate. Residues in the site form hydrogen bonds, hydrophobic interactions. In order to function, the site needs to be in a specific conformation. A tighter fit between a site and the substrate molecule is believed to increase efficiency of a reaction. Most enzymes have deeply buried active sites, which can be accessed by a substrate via access channels, there are two proposed models of how enzymes fit to their specific substrate, the lock and key model and the induced fit model. Emil Fischers lock and key model assumes that the site is a perfect fit for a specific substrate. Daniel Koshlands theory of enzyme-substrate binding is that the active site, the induced fit model is a development of the lock-and-key model and assumes that an active site is flexible and it changes shape until the substrate is completely bound. The substrate is thought to induce a change in the shape of the active site, the hypothesis also predicts that the presence of certain residues in the active site will encourage the enzyme to locate the correct substrate. Conformational changes may occur as the substrate is bound. After the products of the move away from the enzyme. Once the substrate is bound and oriented in the active site, the residues of the catalytic site are typically very close to the binding site, and some residues can have dual-roles in both binding and catalysis. Catalytic residues of the site interact with the substrate to lower the energy of a reaction. They do this by a number of different mechanisms, firstly, they can act as donors or acceptors of protons or other groups on the substrate to facilitate the reaction. They can also form electrostatic interactions to stabilise charge buildup on the state or leaving group

27.
Catalytic triad
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A catalytic triad refers to the three amino acid residues that function together at the centre of the active site of some hydrolase and transferase enzymes. An Acid-Base-Nucleophile triad is a motif for generating a nucleophilic residue for covalent catalysis. The nucleophile is most commonly a serine or cysteine amino acid, as well as divergent evolution of function, catalytic triads show some of the best examples of convergent evolution. Chemical constraints on catalysis have led to the same solution independently evolving in at least 23 separate superfamilies. Their mechanism of action is one of the best studied in biochemistry. The enzymes trypsin and chymotrypsin were first purified in the 1930s, a serine in each of trypsin and chymotrypsin was identified as the catalytic nucleophile in the 1950s. The structure of chymotrypsin was solved by X-ray crystallography in the 1960s, other proteases were sequenced and aligned to reveal a family of related proteases, now called the S1 family. Simultaneously, the structures of the evolutionarily unrelated papain and subtilisin proteases were found to contain analogous triads, the charge-relay mechanism for the activation of the nucleophile by the other triad members was proposed in the late 1960s. As more protease structures were solved by X-ray crystallography in the 1970s and 80s, understanding how chemical constraints on evolution led to the convergence of so many enzyme families on the same triad geometries has developed in the 2010s. The massive body of work on the charge-relay, covalent catalysis used by catalytic triads has led to the mechanism being the best characterised in all of biochemistry. Enzymes that contain an catalytic triad use it for one of two types, either to split a substrate or to transfer one portion of a substrate over to a second substrate. Triads are an inter-dependent set of residues in the site of an enzyme. These triad residues act together to make the nucleophile member highly reactive, catalytic triads perform covalent catalysis using a residue as a nucleophile. The reactivity of the residue is increased by the functional groups of the other triad members. The nucleophile is polarised and oriented by the base, which is itself bound, catalysis is performed in two stages. First, the nucleophile attacks the carbonyl carbon and forces the carbonyl oxygen to accept an electron. The build-up of negative charge on this intermediate is stabilized by an oxanion hole within the active site. The intermediate then collapses back to a carbonyl, ejecting the first half of the substrate, the ejection of this first leaving group is often aided by donation of a proton by the base

28.
Oxyanion hole
–
An oxyanion hole is a pocket in the active site of an enzyme that stabilizes transition state negative charge on a deprotonated oxygen or alkoxide. The pocket typically consists of backbone amides or positively charged residues, stabilising the transition state lowers the activation energy necessary for the reaction, and so promotes catalysis. Additionally, it may allow for insertion or positioning of a substrate, enzymes that catalyse multi-step reactions can have multiple oxyanion holes that stabilise different transition states in the reaction. Enzyme catalysis Active site Transition state Serine proteases#Catalytic mechanism Albert Lehninger, et al

29.
Enzyme promiscuity
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Enzyme promiscuity is the ability of an enzyme to catalyse a fortuitous side reaction in addition to its main reaction. Although enzymes are remarkably specific catalysts, they can often perform side reactions in addition to their main and these promiscuous activities are usually slow relative to the main activity and are under neutral selection. An example of this is the atrazine chlorohydrolase from Pseudomonas sp, ADP which evolved from melamine deaminase, which has very small promiscuous activity towards atrazine, a man-made chemical. Enzymes are evolved to catalyse a reaction on a particular substrate with a high catalytic efficiency. Several theoretical models exist to predict the order of duplication and specialisation events, on the other, enzymes may evolve an increased secondary activity with little loss to the primary activity with little adaptive conflict. A study of three distinct hydrolases has shown the main activity is robust towards change, whereas the activities are more plastic. Specifically, selecting for an activity that is not the activity, does not initially diminish the main activity. The most recent and most clear cut example of evolution is the rise of bioremediating enzymes in the past 60 years. Due to the low number of amino acid changes, these provide an excellent model to investigate enzyme evolution in nature. This issue can be resolved thanks to ancestral reconstruction and this variability in ancestral specificity has not only been observed between different genes, but also within the same gene family. Antithetically, the ancestor before the split had a more pronounced isomaltose-like glucosidase activity. Roy Jensen in 1976 theorised that primordial enzymes had to be highly promiscuous in order for networks to assemble in a patchwork fashion. This primordial catalytic versatility was later lost in favour of highly catalytic specialised orthologous enzymes, as a consequence, many central-metabolic enzymes have structural homologues that diverged before the last universal common ancestor. Promiscuity is however not only a primordial trait, in fact it is very widespread property in modern genomes, a series of experiments have been conducted to assess the distribution of promiscuous enzyme activities in E. coli. In E. coli 21 out of 104 single-gene knockouts tested could be rescued by overexpressing a noncognate E. coli protein, similarly, overexpressing the ORF collection allowed E. coli to gain over an order of magnitude in resistance in 86 out 237 toxic environment. Homologues are sometimes known to display promiscuity towards each others main reactions, despite the divergence the homologues have a varying degree of reciprocal promiscuity, the differences in promiscuity are due to mechanisms involved, particularly the intermediate required. Examples of these are enzymes for primary and secondary metabolism in plants, a promiscuous activity is a non-native activity the enzyme did not evolve to do, but arises due to an accommodating conformation of the active site. When the specificity of enzyme was probed, it was found that it was selective against natural amino acids that were not phenylalanine

30.
Diffusion limited enzyme
–
A Diffusion limited enzyme is an enzyme which catalyses a reaction so efficiently that the rate limiting step is that of substrate diffusion into the active site, or product diffusion out. This is also known as kinetic perfection or catalytic perfection, since the rate of catalysis of such enzymes is set by the diffusion-controlled reaction, it therefore represents an intrinsic, physical constraint on evolution. Diffusion limited perfect enzymes are very rare, most enzymes catalyse their reactions to a rate that is 1, 000-10,000 times slower than this limit. This is due to both the limitations of difficult reactions, and the evolutionary limitations that such high reaction rates do not confer any extra fitness. The theory of diffusion-controlled reaction was utilized by R. A. Alberty, Gordon Hammes, and Manfred Eigen to estimate the upper limit of enzyme-substrate reaction, according to their estimation, the upper limit of enzyme-substrate reaction was 109 M−1 s−1. To address such a paradox, Prof, the new upper limit found by Chou et al. for enzyme-substrate reaction was further discussed and analyzed by a series of follow-up studies. Kinetically perfect enzymes have a specificity constant, kcat/Km, on the order of 108 to 109 M−1 s−1, the rate of the enzyme-catalysed reaction is limited by diffusion and so the enzyme processes the substrate well before it encounters another molecule. Some enzymes operate with kinetics which are faster than diffusion rates, several mechanisms have been invoked to explain this phenomenon. Some proteins are believed to accelerate catalysis by drawing their substrate in, some invoke a quantum-mechanical tunneling explanation whereby a proton or an electron can tunnel through activation barriers, although proton tunneling remains a somewhat controversial idea. It is worth noting that there are not many kinetically perfect enzymes and this can be explained in terms of natural selection. An increase in speed may be favoured as it could confer some advantage to the organism. However, when the catalytic speed outstrips diffusion speed there is no advantage to increase the speed even further. The diffusion limit represents a physical constraint on evolution. Increasing the catalytic speed past the speed will not aid the organism in any way

31.
Cofactor (biochemistry)
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A cofactor is a non-protein chemical compound or metallic ion that is required for a proteins biological activity to happen. These proteins are enzymes, and cofactors can be considered helper molecules that assist in biochemical transformations. A coenzyme that is tightly or even covalently bound is termed a prosthetic group, the two subcategories under coenzyme are cosubstrates and prosthetic groups. Cosubstrates are transiently bound to the protein and will be released at some point, the prosthetic groups, on the other hand, are bound permanently to the protein. Both of them have the function, which is to facilitate the reaction of enzymes. Additionally, some sources also limit the use of the cofactor to inorganic substances. An inactive enzyme without the cofactor is called an apoenzyme, while the enzyme with cofactor is called a holoenzyme. Some enzymes or enzyme complexes require several cofactors, organic cofactors are often vitamins or made from vitamins. Many contain the nucleotide adenosine monophosphate as part of their structures, such as ATP, coenzyme A, FAD and this common structure may reflect a common evolutionary origin as part of ribozymes in an ancient RNA world. It has been suggested that the AMP part of the molecule can be considered to be a kind of handle by which the enzyme can grasp the coenzyme to switch it between different catalytic centers. Cofactors can be divided into two groups, organic cofactors, such as flavin or heme, and inorganic cofactors, such as the metal ions Mg2+, Cu+, Mn2+. Organic cofactors are sometimes divided into coenzymes and prosthetic groups. The term coenzyme refers specifically to enzymes and, as such, on the other hand, prosthetic group emphasizes the nature of the binding of a cofactor to a protein and, thus, refers to a structural property. Different sources give different definitions of coenzymes, cofactors. It should be noted that terms are often used loosely. However, the author could not arrive at a single all-encompassing definition of a coenzyme, the study of these cofactors falls under the area of bioinorganic chemistry. In nutrition, the list of essential trace elements reflects their role as cofactors, in humans this list commonly includes iron, magnesium, manganese, cobalt, copper, zinc, and molybdenum. Although chromium deficiency causes impaired glucose tolerance, no human enzyme that uses this metal as a cofactor has been identified, iodine is also an essential trace element, but this element is used as part of the structure of thyroid hormones rather than as an enzyme cofactor

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Enzyme catalysis
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Enzyme catalysis is the increase in the rate of a chemical reaction by the active site of a protein. The protein catalyst may be part of a complex, and/or may transiently or permanently associate with a Cofactor. Catalysis of biochemical reactions in the cell is vital due to the very low rates of the uncatalysed reactions at room temperature and pressure. A key driver of protein evolution is the optimization of such catalytic activities via protein dynamics, the mechanism of enzyme catalysis is similar in principle to other types of chemical catalysis. By providing an alternative reaction route the enzyme reduces the required to reach the highest energy transition state of the reaction. The reduction of activation increases the amount of reactant molecules that achieve a sufficient level of energy, such that they reach the activation energy. As with other catalysts, the enzyme is not consumed during the reaction but is recycled such that a single enzyme performs many rounds of catalysis, the favored model for the enzyme-substrate interaction is the induced fit model. The advantages of the induced fit mechanism arise due to the effect of strong enzyme binding. There are two different mechanisms of substrate binding, uniform binding, which has strong binding, and differential binding. The stabilizing effect of uniform binding increases both substrate and transition state binding affinity, while differential binding increases only transition state binding affinity, both are used by enzymes and have been evolutionarily chosen to minimize the activation energy of the reaction. It is important to clarify, however, that the induced fit concept cannot be used to rationalize catalysis and that is, the chemical catalysis is defined as the reduction of Ea‡ relative to Ea‡ in the uncatalyzed reaction in water. The induced fit only suggests that the barrier is lower in the form of the enzyme. Induced fit may be beneficial to the fidelity of molecular recognition in the presence of competition, →→→editor These conformational changes also bring catalytic residues in the active site close to the chemical bonds in the substrate that will be altered in the reaction. After binding takes place, one or more mechanisms of catalysis lowers the energy of the transition state. This effect is analogous to an increase in concentration of the reagents. The binding of the reagents to the enzyme gives the reaction intramolecular character, however, the situation might be more complex, since modern computational studies have established that traditional examples of proximity effects cannot be related directly to enzyme entropic effects. Also, the original proposal has been found to largely overestimate the contribution of orientation entropy to catalysis. Histidine is often the residue involved in these reactions, since it has a pKa close to neutral pH

high resolution crystal structure of a mg2-dependent 5-aminolevulinic acid dehydratase

The PBGS quaternary structure equilibrium includes an inactive hexamer (PDB id 1PV8) that does not have subunit interactions necessary for an ordered active site lid. Dissociation to the pro-hexamer dimer can be followed by a conformational change that reorients the two αβ-barrel domains to form the pro-octamer dimer. Association of pro-octamer dimer to octamer (PDB id 1E51) includes formation of subunit interfaces that support order in the active site lid.

The energies of the stages of a chemical reaction. Uncatalysed (dashed line), substrates need a lot of activation energy to reach a transition state, which then decays into lower-energy products. When enzyme catalysed (solid line), the enzyme binds the substrates (ES), then stabilizes the transition state (ES‡) to reduce the activation energy required to produce products (EP) which are finally released.